1. Field of the Invention
The present invention relates generally to agents and nucleic acid targets for regulating gene expression. More specifically, the present invention relates to compounds, compositions, and methods for regulation of the expression of nucleic acids encoding Bcl-2.
2. Technical Background
The genetic blueprint of mankind is written and stored in the chromosomes found in the nuclei of somatic cells. These chromosomes are each made up of a single molecule of DNA. DNA is a double-stranded polymer compound whose strands are chains of the nucleotide subunits adenine, thymine, cytosine, and guanine. These chains are held to each other by hydrogen bonds formed between complementary nucleotides on the chains. Adenine (A) forms hydrogen bonds with thymine (T); and guanine (G) forms hydrogen bonds with cytosine (C). Contiguous sets of three of these nucleobases in the nucleotide strands code for individual amino acids. Together, the nucleotide chains of DNA code for all of the various proteins needed to build, maintain, and fuel the human body.
Proteins are translated from transcribed copies of DNA called messenger RNA. Messenger RNA is made when DNA partially unwinds, allowing the sequence of the DNA to be copied and transcribed by cellular machinery. Messenger RNA is made of similar components as DNA, with the substitution of uracil for thymine and the substitution of ribose for deoxyribose in all nucleotides. Once the transcription of a messenger RNA is completed, it is transported from the nucleus to the cytosol. There, the mRNA is engaged by the enzymes that are the protein-manufacturing machinery of the cell. The enzymes and other associated molecules making up this machinery bind to the mRNA and begin to interpret the code of the messenger RNA. As they do so, they also assemble and bind the amino acids encoded into chains to produce the protein coded for by the original DNA.
Illnesses and disease states such as some cancers have been linked to the over-, under-, or mis-production of specific proteins. In some of these cases, a defect present in the gene causes the production of messenger RNAs that code for a nonfunctional or inefficient protein. Additionally, some diseases may be controlled by increasing or decreasing the normal production of a protein. Specifically, it is thought that in some cancers, if the gene coding for a specific protein could be regulated, the cancer would abate, or would become more susceptible to treatment. As a result of this, much research has been conducted to find a method of modulating the activity of a gene. In one specific study, it was determined that down-regulation of Bcl-2 production in prostate cancer cells inhibited their growth and rendered them susceptible to adriamycin-induced apoptosis. Shi et al., Cancer Biother. Radiopharm., 16(5):421-9 (October 2001).
One technology used for regulating the expression of a gene is antisense technology. Antisense technology controls the production of a protein by binding a molecule to the nucleic acid coding for the protein. Generally this molecule is a short length of DNA or RNA commonly referred to as an antisense oligonucleotide. These oligonucleotides are complementary to a segment of the nucleic acid. In use, these antisense oligonucleotides are administered to a cell or tissue desired to be treated. The oligonucleotides are taken into the cell where they associate with and bind to a region of the nucleic acid encoding the protein to which they are complementary. This binding prevents normal interaction of the nucleic acid with cellular machinery such as RNA translation enzymes or induces the degradation of the message RNA. This inhibits or prevents proper translation of the message RNA.
Antisense is regarded by many as a powerful technology since the antisense oligonucleotides used may be carefully targeted to specific regions on the nucleic acids, thus preventing interaction of the oligonucleotides with other molecules not desired to be inhibited or activated. These specific regions of the selected nucleic acids are often referred to as target sequences. Because antisense oligonucleotides may be so carefully targeted to these target sequences, antisense oligonucleotides may be used to provide compositions, such as drugs, that have near-absolute specificity, high efficacy, low toxicity, and few side effects.
These benefits are overshadowed, however, by the difficulty involved in locating effective antisense oligonucleotides for use with a gene. Despite the fact that generally there are a large number of potential oligonucleotides available for each individual gene, different antisense oligonucleotides have different effects on gene expression. This has been shown to be due at least in part to the final folded structure of the nucleic acid which may block access to some regions of the nucleic acid sequence, thus rendering some oligonucleotides ineffective. Further, the work of characterizing effective sites must be repeated for each individual gene desired to be targeted. This process is generally a long and expensive one. Many disease states, including cancer, stand to benefit from successful antisense therapies, but oligonucleotides useful with specific genes are elusive.
Bcl-2 is an inner mitochondrial membrane protein that has been shown to block apoptotic cell death in some specific cell types. The expression of Bcl-2 has been shown to be involved in apoptosis in the thymus (Kanavaros et al., Histol. Histopathol. 16(4):1005-12 (October 2001). Bcl-2 has also been shown to play a role in prostatic cancers, and has been specifically linked to aggressive tumors common in specific racial groups. Shi et al., Cancer Biother. Radiopharm., 16(5):421-9 (October 2001); Slothower, Study Suggests Bcl-2 Gene as a Cause for Aggressive Prostate Cancer in African American Men, U.C. Davis Med. Ctr., (May 1998). As briefly noted above, a study in which Bcl-2 production was down-regulated in prostate cancer cells showed inhibition of cell growth and increased sensitivity to treatments designed to induce apoptosis. Shi et al., Biother. Radiopharm., 16(5):421-9 (October 2001).
Accordingly, it would be an advancement in the art to provide inhibitory oligonucleotide compounds configured to bind to, and consequently, to modulate the activity of nucleic acids encoding proteins which play a role in diseases such as cancer. It would be a further advancement to provide effective target sites for Bcl-2 gene regulation. It would be an advancement in the art to provide oligonucleotides complementary to effective antisense target regions of a nucleic acid encoding Bcl-2. It would be a further benefit in the art to provide compositions such as medications, including such oligonucleotides. Finally, it would be an improvement in the art to provide methods of using such oligonucleotides and compositions.
Such oligonucleotides, target regions, compositions including such oligonucleotides, and methods of their use are disclosed herein.